André Schütte

The mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system

The gastrointestinal tract is covered by mucus that has different properties in the stomach, small intestine, and colon. The large highly glycosylated gel‐forming mucins MUC2 and MUC5AC are the major components of the mucus in the intestine and stomach, respectively. In the small intestine, mucus limits the number of bacteria that can reach the epithelium and the Peyers patches. In the large intestine, the inner mucus layer separates the commensal bacteria from the host epithelium. The outer colonic mucus layer is the natural habitat for the commensal bacteria. The intestinal goblet cells secrete not only the MUC2 mucin but also a number of typical mucus components: CLCA1, FCGBP, AGR2, ZG16, and TFF3. The goblet cells have recently been shown to have a novel gate‐keeping role for the presentation of oral antigens to the immune system. Goblet cells deliver small intestinal luminal material to the lamina propria dendritic cells of the tolerogenic CD103+ type. In addition to the gel‐forming mucins, the transmembrane mucins MUC3, MUC12, and MUC17 form the enterocyte glycocalyx that can reach about a micrometer out from the brush border. The MUC17 mucin can shuttle from a surface to an intracellular vesicle localization, suggesting that enterocytes might control and report epithelial microbial challenge. There is communication not only from the epithelial cells to the immune system but also in the opposite direction. One example of this is IL10 that can affect and improve the properties of the inner colonic mucus layer. The mucus and epithelial cells of the gastrointestinal tract are the primary gate keepers and controllers of bacterial interactions with the host immune system, but our understanding of this relationship is still in its infancy.

Composition and functional role of the mucus layers in the intestine

In discussions on intestinal protection, the protective capacity of mucus has not been very much considered. The progress in the last years in understanding the molecular nature of mucins, the main building blocks of mucus, has, however, changed this. The intestinal enterocytes have their apical surfaces covered by transmembrane mucins and the whole intestinal surface is further covered by mucus, built around the gel-forming mucin MUC2. The mucus of the small intestine has only one layer, whereas the large intestine has a two-layered mucus where the inner, attached layer has a protective function for the intestine, as it is impermeable to the luminal bacteria.

The composition of the gut microbiota shapes the colon mucus barrier

Two C57BL/6 mice colonies maintained in two rooms of the same specific pathogen‐free (SPF) facility were found to have different gut microbiota and a mucus phenotype that was specific for each colony. The thickness and growth of the colon mucus were similar in the two colonies. However, one colony had mucus that was impenetrable to bacteria or beads the size of bacteria—which is comparable to what we observed in free‐living wild mice—whereas the other colony had an inner mucus layer penetrable to bacteria and beads. The different properties of the mucus depended on the microbiota, as they were transmissible by transfer of caecal microbiota to germ‐free mice. Mice with an impenetrable mucus layer had increased amounts of Erysipelotrichi, whereas mice with a penetrable mucus layer had higher levels of Proteobacteria and TM7 bacteria in the distal colon mucus. Thus, our study shows that bacteria and their community structure affect mucus barrier properties in ways that can have implications for health and disease. It also highlights that genetically identical animals housed in the same facility can have rather distinct microbiotas and barrier structures.

Studies of mucus in mouse stomach, small intestine, and colon. I. Gastrointestinal mucus layers have different properties depending on location as well as over the Peyer's patches

Colon has been shown to have a two-layered mucus system where the inner layer is devoid of bacteria. However, a complete overview of the mouse gastrointestinal mucus system is lacking. We now characterize mucus release, thickness, growth over time, adhesive properties, and penetrability to fluorescent beads from stomach to distal colon. Colon displayed spontaneous mucus release and all regions released mucus in response to carbachol and PGE2, except the distal colon and domes of Peyers patches. Stomach and colon had an inner mucus layer that was adherent to the epithelium. In contrast, the small intestine and Peyers patches had a single mucus layer that was easily aspirated. The inner mucus layer of the distal colon was not penetrable to beads the size of bacteria and the inner layer of the proximal colon was only partly penetrable. In contrast, the inner mucus layer of stomach was fully penetrable, as was the small intestinal mucus. This suggests a functional organization of the intestinal mucus system, where the small intestine has loose and penetrable mucus that may allow easy penetration of nutrients, in contrast to the stomach, where the mucus provides physical protection, and the colon, where the mucus separates bacteria from the epithelium. This knowledge of the mucus system and its organization improves our understanding of the gastrointestinal tract physiology.

Proteomic Analyses Reveal an Acidic Prime Side Specificity for the Astacin Metalloprotease Family Reflected by Physiological Substrates

Astacins are secreted and membrane-bound metalloproteases with clear associations to many important pathological and physiological processes. Yet with only a few substrates described their biological roles are enigmatic. Moreover, the lack of knowledge of astacin cleavage site specificities hampers assay and drug development. Using PICS (proteomic identification of protease cleavage site specificity) and TAILS (terminal amine isotopic labeling of substrates) degradomics approaches >3000 cleavage sites were proteomically identified for five different astacins. Such broad coverage enables family-wide determination of specificities N- and C-terminal to the scissile peptide bond. Remarkably, meprin α, meprin β, and LAST_MAM proteases exhibit a strong preference for aspartate in the peptide (P)1′ position because of a conserved positively charged residue in the active cleft subsite (S)1′. This unparalleled specificity has not been found for other families of extracellular proteases. Interestingly, cleavage specificity is also strongly influenced by proline in P2′ or P3′ leading to a rare example of subsite cooperativity. This specificity characterizes the astacins as unique contributors to extracellular proteolysis that is corroborated by known cleavage sites in procollagen I+III, VEGF (vascular endothelial growth factor)-A, IL (interleukin)-1β, and pro-kallikrein 7. Indeed, cleavage sites in VEGF-A and pro-kallikrein 7 identified by terminal amine isotopic labeling of substrates matched those reported by Edman degradation. Moreover, the novel substrate FGF-19 was validated biochemically and shown to exhibit altered biological activity after meprin processing.

Normalization of Host Intestinal Mucus Layers Requires Long-Term Microbial Colonization

The intestinal mucus layer provides a barrier limiting bacterial contact with the underlying epithelium. Mucus structure is shaped by intestinal location and the microbiota. To understand how commensals modulate gut mucus, we examined mucus properties under germ-free (GF) conditions and during microbial colonization. Although the colon mucus organization of GF mice was similar to that of conventionally raised (Convr) mice, the GF inner mucus layer was penetrable to bacteria-sized beads. During colonization, in which GF mice were gavaged with Convr microbiota, the small intestine mucus required 5 weeks to be normally detached and colonic inner mucus 6 weeks to become impenetrable. The composition of the small intestinal microbiota during colonization was similar to Convr donors until 3 weeks, when Bacteroides increased, Firmicutes decreased, and segmented filamentous bacteria became undetectable. These findings highlight the dynamics of mucus layer development and indicate that studies of mature microbe-mucus interactions should be conducted weeks after colonization.

Microbial-induced meprin β cleavage in MUC2 mucin and a functional CFTR channel are required to release anchored small intestinal mucus.

Significance Mucus with its major constituent, the gel-forming mucins, is important for protecting the host epithelium from bacteria. Under normal conditions, these mucin networks are constantly released into the small intestinal lumen. This release required a proteolytic cleavage in the mucin by the metalloprotease meprin β and was absent in germ-free animals but induced by bacteria. The small intestinal mucus in cystic fibrosis is also attached, not due to lack of enzyme, but rather that the mucin is not properly unfolded in the absence of a functional cystic fibrosis transmembrane conductance regulator channel and sufficient bicarbonate levels. Mucus can thus appear both attached and released as part of a system controlling bacterial removal. This new concept may lead to new ways for the treatment and therapy of cystic fibrosis. The mucus that covers and protects the epithelium of the intestine is built around its major structural component, the gel-forming MUC2 mucin. The gel-forming mucins have traditionally been assumed to be secreted as nonattached. The colon has a two-layered mucus system where the inner mucus is attached to the epithelium, whereas the small intestine normally has a nonattached mucus. However, the mucus of the small intestine of meprin β-deficient mice was now found to be attached. Meprin β is an endogenous zinc-dependent metalloprotease now shown to cleave the N-terminal region of the MUC2 mucin at two specific sites. When recombinant meprin β was added to the attached mucus of meprin β-deficient mice, the mucus was detached from the epithelium. Similar to meprin β-deficient mice, germ-free mice have attached mucus as they did not shed the membrane-anchored meprin β into the luminal mucus. The ileal mucus of cystic fibrosis (CF) mice with a nonfunctional cystic fibrosis transmembrane conductance regulator (CFTR) channel was recently shown to be attached to the epithelium. Addition of recombinant meprin β to CF mucus did not release the mucus, but further addition of bicarbonate rendered the CF mucus normal, suggesting that MUC2 unfolding exposed the meprin β cleavage sites. Mucus is thus secreted attached to the goblet cells and requires an enzyme, meprin β in the small intestine, to be detached and released into the intestinal lumen. This process regulates mucus properties, can be triggered by bacterial contact, and is nonfunctional in CF due to poor mucin unfolding.

Studies of mucus in mouse stomach, small intestine, and colon. II. Gastrointestinal mucus proteome reveals Muc2 and Muc5ac accompanied by a set of core proteins

The mucus that protects the surface of the gastrointestinal tract is rich in specialized O-glycoproteins called mucins, but little is known about other mucus proteins or their variability along the gastrointestinal tract. To ensure that only mucus was analyzed, we combined collection from explant tissues mounted in perfusion chambers, liquid sample preparation, single-shot mass spectrometry, and specific bioinformatics tools, to characterize the proteome of the murine mucus from stomach to distal colon. With our approach, we identified ∼1,300 proteins in the mucus. We found no differences in the protein composition or abundance between sexes, but there were clear differences in mucus along the tract. Noticeably, mucus from duodenum showed similarities to the stomach, probably reflecting the normal distal transport. Qualitatively, there were, however, fewer differences than might had been anticipated, suggesting a relatively stable core proteome (∼80% of the total proteins identified). Quantitatively, we found significant differences (∼40% of the proteins) that could reflect mucus specialization throughout the gastrointestinal tract. Hierarchical clustering pinpointed a number of such proteins that correlated with Muc2 (e.g., Clca1, Zg16, Klk1). This study provides a deeper knowledge of the gastrointestinal mucus proteome that will be important in further understanding this poorly studied mucosal protection system.

Let It Flow: Morpholino Knockdown in Zebrafish Embryos Reveals a Pro-Angiogenic Effect of the Metalloprotease Meprin α2

Background Meprin metalloproteases are thought to be involved in basic physiological functions such as cell proliferation and tissue differentiation. However, the specific functions of these enzymes are still ambiguous, although a variety of growth factors and structural proteins have been identified as meprin substrates. The discovery of meprins α1, α2 and β in teleost fish provided the basis for uncovering their physiological functions by gene silencing in vivo. Methodology/Principal Findings A Morpholino knockdown in zebrafish embryos targeting meprin α1 and β mRNA caused defects in general tissue differentiation. But meprin α2 morphants were affected more specifically and showed severe failures in the formation of the vascular system provoking the hypothesis of a pro-angiogenic effect. The blood circulation was largely diminished resulting in erythrocyte accumulation. These phenotypes mimic a previously described VEGF-A morphant, revealing a possible role of meprin α in VEGF-A activation. Indeed, human recombinant meprin α processed the vascular endothelial growth factor-A (VEGF-A) specifically, revealing the same cleavage products detectable for VEGF from zebrafish whole lysate. Conclusions/Significance Our results demonstrate that meprin metalloproteases are important for cell differentiation and proliferation already during embryogenesis, predominantly by the activation of growth factors. Thus, we conclude that meprins play a significant role in VEGF-A processing, subsequently regulating angiogenesis. Therefore, meprin α might be a new therapeutic target in cardiovascular diseases or in tumor growth inhibition.

News from an Ancient World: Two Novel Astacin Metalloproteases from the Horseshoe Crab

In this work, we report the cloning, heterologous expression, and characterization of two novel astacin proteases from the chelicerate Limulus polyphemus (horseshoe crab), designated as LAST (Limulus astacin) and LAST_MAM (Limulus astacin containing a MAM domain), respectively. The expression pattern showed ubiquitous occurrence of LAST_MAM, while LAST was predominantly restricted to the eyes and brain, indicating a function in the nervous system. Both enzymes contain the characteristic metzincin-type zinc-binding region and Met turn. While LAST is made up only of the typical prodomain and astacin-like protease domain, LAST_MAM contains an additional MAM (meprin A5 protein tyrosine phosphatase micro) domain, which so far only has been found in few astacins such as the vertebrate meprin Hydra and squid enzymes, and in a number of other extracellular proteins such as A5 protein and tyrosine phosphatase micro. These gave rise to the designation MAM for this protein module. MAM domains have been shown to be responsible for protein oligomerization in meprin proteases and tyrosine phosphatase micro. Since the horseshoe crab has kept its body plan for almost half a billion years, it is therefore a privileged organism for the study of protease evolution. In this context, we could show by phylogenetic analysis that this protease is not related to the other MAM-domain-containing astacins indicating different evolutionary origins of these proteins. Moreover, we clearly demonstrated the divergent evolvement of the MAM module itself, and not only with regard to proteases. However, there are some unique functional features that are not shared by other members of this protein family. For example, LAST_MAM is the only astacin protease known so far that is active in its zymogen form, indicating that the presence of the N-terminal propeptide does not prevent proteolytic activity.